151 TAP polymorphisms in leprosy 6. ASSOCIATION OF TAP1 AND TAP2 POLYMORPHISMS IN LEPROSY INTRODUCTION Leprosy caused by Mycobacterium leprae primarily affects peripheral nerves and skin. Disease presents as spectrum and is governed by the host immune response. In an endemic pocket, on exposure to M. leprae, majority of the population do not develop overt clinical disease indicating the involvement of host immunity in disease development (Martelli et al 2000, Siddiqui et al 2001, Kumar et al 2004, Ogunbiyi et al 2004, Meima et al 2004, WHO Weekly Epidemiological Report, 2007). Thus, outcome of leprosy is in part due to host genes that controls the initial infection and the host immune response (Scollard et al 2006). A substantial body of evidence suggests that multi factorial genetic factors control susceptibility of humans to infection by M. leprae (Jamieson et al 2004, Mira et al 2004, Alter et al 2008, Da Silva et al 2009). Evidence for the role of host genetics in leprosy or the type of disease manifestation was obtained from epidemiological segregation and twin studies (Ishii et al 1993). Candidate gene analysis and genome wide studies have implicated various genes and regions on chromosomes in susceptibility or resistance towards the infection (Miller et al 2004, Zhang et al 2009) One of the first candidate gene studied in leprosy was Transporter Associated with antigen presentation (TAP) (Rajalingam et al 1997). Two linkage
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151 TAP polymorphisms in leprosy
6. ASSOCIATION OF TAP1 AND TAP2 POLYMORPHISMS
IN LEPROSY
INTRODUCTION
Leprosy caused by Mycobacterium leprae primarily affects peripheral
nerves and skin. Disease presents as spectrum and is governed by the
host immune response. In an endemic pocket, on exposure to M.
leprae, majority of the population do not develop overt clinical disease
indicating the involvement of host immunity in disease development
(Martelli et al 2000, Siddiqui et al 2001, Kumar et al 2004, Ogunbiyi
et al 2004, Meima et al 2004, WHO Weekly Epidemiological Report,
2007). Thus, outcome of leprosy is in part due to host genes that
controls the initial infection and the host immune response (Scollard
et al 2006). A substantial body of evidence suggests that multi
factorial genetic factors control susceptibility of humans to infection
by M. leprae (Jamieson et al 2004, Mira et al 2004, Alter et al 2008,
Da Silva et al 2009). Evidence for the role of host genetics in leprosy or
the type of disease manifestation was obtained from epidemiological
segregation and twin studies (Ishii et al 1993). Candidate gene
analysis and genome wide studies have implicated various genes and
regions on chromosomes in susceptibility or resistance towards the
infection (Miller et al 2004, Zhang et al 2009) One of the first
candidate gene studied in leprosy was Transporter Associated with
antigen presentation (TAP) (Rajalingam et al 1997). Two linkage
152 TAP polymorphisms in leprosy
studies have associated chromosome 6pq21 with leprosy susceptibility
(Abel et al 2008; Mira et al 2003).
TAP is a member of the family of ABC transporters that play a key role
in the processing and presentation of endogenous antigen to CD8+ T
cells by MHC class I pathway. In contrast, MHC class II restricted
antigens are generated by endosomal proteases and presented to
CD4+ T cells. Yet, exogenous antigens can be channeled via the
endogenous pathway where antigen presentation is mediated by MHC
class I molecules (Robinson et al 2002). Thus, the ER could be a site
for loading newly synthesized MHC class II molecules and it may play
a dual role in both endogenous and exogenous antigen processing
(Tewari et al 2005).
TAP is made up of TAP1 and TAP2 polypeptides which consist of 11
and 9 exons respectively. The significance of TAP proteins for antigen
presentation to CD8+ T cells has been demonstrated both in cell lines
and in knockout mice. Restoration of TAP1 and TAP2 in cell lines
deficient in these molecules completely restored the ability of these
cells to present antigen to T lymphocytes (Van Kaer et al 1992). It is
possible that polymorphisms in the TAP gene can influence the
antigen peptide selection, transport process and alter immune
response regulation.
153 TAP polymorphisms in leprosy
Over 13 polymorphic sites have been reported in the TAP1 gene
(Colona et al 1992, Powis et al 1993, Shi et al 1997, Tang et al 2001,
Lajoie et al 2003). The most commonly reported polymorphic residues
are at positions 333 and 637 (TAP1), 565 and 665 (TAP2). Because of
their endogenous antigen presentation and location on MHC, TAP
genes have been known to be important candidate genes in
autoimmune diseases and polymorphisms have been implicated in
multiple sclerosis, rheumatoid arthritis, allergic rhinitis and Insulin
dependent diabetes mellitus (Middleton et al 1994, Singal et al 1994,
Cailit- Zuchman et al 1995, Ishihara et al 1996, Foley et al 1999, Kim
et al 2007, Feng et al 2009).
Mycobacterial infectious disease like leprosy and tuberculosis are
known to associated with HLA-DR2 and its subtypes HLA DRB 1501
and 1502 ( Mehra et al 1992, Mehra et al 1995, Rajalingam et al
1996) . The TAP gene is located between HLA-DP and HLA-DQ of
chromosome 6 which has been implicated in susceptibility to leprosy
(Mira et al 2003, Zhang et al 2009). There is dearth in information of
TAP polymorphisms in South Indian population, hence we embarked
upon this study to elucidate the association of TAP1 and TAP2
polymorphisms in leprosy.
154 TAP polymorphisms in leprosy
6.1 Methodology
6.1.1 Study Subjects:
Leprosy patients (n=100) reporting to two LEPRA projects: BPHRC,
HYLEP, and Sivananda Rehabilitation home were enrolled in the study
after obtaining informed written consent. All patients had slit skin
smear examination and skin lesion was evaluated by histopathology.
Out of the total 100 patients, 71 were classified as lepromatous and
29 as tuberculoid. Control subjects were endemic healthy individuals
who belonged to the similar socio-economic status and ethnic
background. None of them had any family history of leprosy,
tuberculosis or other major diseases. The study was approved by the
Institutional Ethical Committee (IEC).
6.1.2 DNA Extraction:
DNA was extracted from the biopsies using Qiagen DNeasy kit
according to the manufacturer’s instructions as mentioned in
materials and methods (Cat No 69506). DNA thus obtained was
subjected to tetra primer Amplification Refractory Mutation System
(ARMS) PCR amplification using previously published primers (Powis
et al 1993) for the TAP1 and TAP2 gene as described in chapter
materials and methods (3.12)
155 TAP polymorphisms in leprosy
6.1.3 DNA amplification using ARMS-PCR:
TAP1 and TAP2 genes were amplified using a tetra primer
Amplification Refraction Mutation System-PCR (ARMS-PCR) as
described earlier (Newton et al 1989) as described in materials and
methods (3.15). The protocol was further standardized with
modifications in annealing temperatures. Polymorphic residues at
codon 333 and 637 for TAP1, 565 and 665 for TAP2 were analyzed. A
set of four primers were used for each polymorphic site. Sequencing
using ABI 3130 for a few representative samples for the detection of
polymorphic residues was outsourced from a local facility.
6.1.4 Statistical analysis:
All statistical analyses were performed with SPSS and Graph Pad
Prism ((version 5) IL, USA). A chi-square test was used to compare the
distribution of TAP1 and TAP2 genotypes between control and leprosy
groups. Odds ratios (OR) with a 95% confidence interval (CI) were
calculated using the Fisher’s exact chi-square test. Differences were
considered significant if p<0.05. Hardy-Weinberg law was used to
analyze all the possible genotype frequencies and according to the
classical method to assess if the system is in genetic equilibrium and
if there was a significant excess of homozygosity. Haplotype analysis
were performed using the Haploview v4.2 software (Barrett et al, 2005)
156 TAP polymorphisms in leprosy
6.2 Results
6.2.1 TAP1 333 Isoleucine to Valine polymorphism:
The heterozygous genotype (Isoleucine (A)/Valine (G)) was positively
associated with leprosy (71%) when compared with controls (46%)
(p<0.0005). The frequency of AA genotype was significantly low in
patients (27%) when compared to controls (50%) presenting negative
association of this genotype with the disease group (p<0.001).
Frequency of GG homozygous genotype was observed to be similar in
patients and controls with 2% and 4% respectively. G allele was
positively associated with the disease when compared A allele (Table
6.1). Significance was not observed across leprosy spectrum for TAP1
333 (Table 6.2). The genotype frequencies of controls were not found
to be in Hardy-Weinberg equilibrium.
PCR products when resolved on 2% agarose gel electrophoresis
showed internal control band of 533bp, Valine specific band of 351bp
and Isoleucine specific band of 241bp. Hence the presence of two
bands of 351 and 241 basepairs in a single lane along with the 533 bp
control indicates Isoleucine/Valine heterozygote, while a 351 bp or
241 bp amplicon indicates homozygosity for Valine or Isoleucine
respectively (Figure 6.1).
157 TAP polymorphisms in leprosy
Figure 6.1: ARMS PCR for TAP1 gene position 333
A representative agarose gel (2%) picture showing amplification
products for TAP1 gene at position 333.
Lanes 1,3, 5,7 and 8 - heterozygous for Isoleucine/Valine (AG).
Lanes 2, 4 and 6 - homozygous for Isoleucine (AA).
Lane 5 - homozygous for Valine (GG).
Lane 9 - 100 bp molecular marker.
Internal control (533bp) is common for all PCR products.
158 TAP polymorphisms in leprosy
Fig 6.2: Chromatograms showing genotype of TAP1333
A
B
C
Partial chromatograms of TAP1 gene at position 333 showing peaks
A: Homozygous for Isoleucine ATC; B: Homozygous for Valine
C: Heterozygous for Isoleucine/Valine as indicated by overlap of A
and G peaks.
159 TAP polymorphisms in leprosy
Table 6.1: Distribution of TAP1 (333) genotypes and Allele in leprosy
and control group
Table 6.2: Distribution of TAP1 (333) genotypes and alleles across
leprosy spectrum.
Using Fisher’s exact test the distribution of TAP1 333 genotypes and
alleles was compared. % represents the percentage frequency of
genotype; *p-value<0.05 was considered significant; OR: Odds Ratio;
CI: Confidence intervals.
Genotype
/ Alleles
Amino
acid
Leprosy
(100)
n(%)
Control
(100)
n(%)
Odds
ratio
(OR)
95% CI p value
TAP1333
Genotype
AA Iso/Iso 27(27) 50(50) 0.36 0.20-0.66 0.001*
AG Iso/Val 71(71) 46(46) 2.87 1.60-5.15 0.0005*
GG Val/Val 2(2) 4(4) 0.48 0.08-2.73 NS
Allele
A Iso 125 146 0.61 0.40-0.94 0.032*
G Val 75 54 1.62 1.0-2.47 0.032*
Genotype
/ Alleles
Amino
acid
BL+LL
(71)
n(%)
BT
(29)
n(%)
Odds
ratio
(OR)
95% CI p value
TAP1 333
Genotype
AA Iso/Iso 21(27.6) 8(27.6) 1.1 0.38-3.34 0.99
AG Iso/Val 48(71.6) 21(72.4) 0.79 0.26-2.23 0.82
GG Val/Val 2 0
Allele
A Iso 88 37 0.99 0.52-1.89 0.99
G Val 50 21 0.99 0.52-1.89 0.99
160 TAP polymorphisms in leprosy
6.2.2 TAP1 637 to Aspartate Glycine (A to G) polymorphism
A significantly higher frequency of the heterozygous genotype (Aspartic
Acid (A)/Glycine (G)) (54%) was observed in patients when compared
to controls (34%). The frequency of AA genotype was significantly low
in patients (45%) when compared to controls (60%). On the other
hand the frequency of GG homozygous was observed to be similar in
patients (1%) and controls (6%) respectively. G allele was positively
associated with the disease when compared A allele (Table 6.3). No
significance was observed across leprosy spectrum for TAP1 637
(Table 6.4). The genotype frequencies of controls were not found to be
in Hardy-Weinberg equilibrium.
PCR products when resolved on 2% agarose gel electrophoresis
showed control band of 429bp, Aspartic Acid specific band of 380bp
and Glycine specific band of 180bp. Hence the presence of two bands
of 380 and 180 basepairs in a single lane along with the 429 bp
control indicates Aspartic Acid/glycine heterozygote, while a 380 bp or
180 bp amplicon indicates homozygosity for Aspartic Acid or Glycine
respectively (Figure 6.3).
161 TAP polymorphisms in leprosy
Figure 6.3: ARMS PCR for TAP1 gene position 637
A representative agarose gel (2%) picture showing amplification
products for TAP1 gene at position 637.
Lanes 3, 4 and 10 - heterozygous for Aspartic Acid/Glycine (AG)
Lanes 2,5,6,7,8,9 and 10 - homozygous for Aspartic Acid (AA)
Lane 9 is a 100 bp molecular marker
Internal control (429bp) is common for all PCR products.
162 TAP polymorphisms in leprosy
Fig 6.4: Chromatograms showing genotype of TAP1637
A
B
C
Partial chromatograms of TAP1 at position 637 showing peaks
A: Homozygous for Aspartic Acid ; B:Homozygous for Glycine
C: Heterozygous for Aspsartic Acid/Glycine as indicated by overlap of
A and G peaks
163 TAP polymorphisms in leprosy
Table 6.3: Distribution of TAP1 (637) genotypes and alleles in leprosy
patients and control group
Table 6.4: Distribution of TAP1 (637) genotype and alleles across
leprosy spectrum
Using Fisher’s exact test the distribution of TAP1 637 genotypes and
alleles was compared. % represents the percentage frequency of
genotype; *p-value<0.05 was considered significant; OR: Odds Ratio;
CI: Confidence intervals.
Genotype
/ Alleles
Amino
acid
Leprosy
(100)
n(%)
Control
(100)
n(%)
Odds ratio
(OR)
95% CI p value
TAP1 637
Genotype
AA Asp/Asp 45(45) 60(60) 0.54 0.31-0.95 0.047*
AG Asp/Gly 54(54) 34(34) 2.27 1.28-4.03 0.006*
GG Gly/Gly 1(1) 6(6) 0.15 0.01-1.33 0.11
Allele
A Asp 144 154 0.68 0.40-1.08 0.13
G Gly 56 46 1.47 0.92-2.37 0.13
Genotype /
Alleles
Amino
acid
BL+LL
(71)
n(%)
BT (29)
n(%)
Odds
ratio
(OR)
95% CI p value
TAP1 637
Genotype
AA Asp/Asp 36(50.7) 10(34.5) 1.9 0.79-4.78 0.18
AG Asp/Gly 34(47.9) 19(65.5) 0.48 0.19-1.18 0.12
GG Gly/Gly 1(1.4) 0
Allele
A Asp 106 39 1.4 0.73-2.79 0.3
G Gly 36 19 0.69 0.35-1.35 0.3
164 TAP polymorphisms in leprosy
6.2.3 TAP2 565 Alanine to Threonine (G to A) polymorphism:
The frequency of homozygous genotype (Alanine (G)/Alanine (G)) was
similar in patients (95%) and controls (97%). Frequency of
heterozygous GA genotype had no significant difference between
patient (3%) and control (4%) (Table 6.5). Significant association was
not seen for TAP2 565 between the leprosy spectrum (Table 6.6). The
genotype frequencies did not follow Hardy-Weinberg equilibrium
PCR products when resolved on 2% agarose gel electrophoresis
showed control band of 400bp, Alanine specific band of 298bp and
Threonine specific band of 161bp. Hence the presence of two bands of
298 and 161 base pairs in a single lane along with the 400 bp control
indicates Alanine/Threonine (GA) heterozygote, while a 298 bp or 161
bp amplicon indicates homozygosity for Alanine(GG) or Threonine(AA)
respectively(Figure 6.5)
165 TAP polymorphisms in leprosy
Fig 6.5. ARMS PCR for TAP1 gene position 565
A representative agarose gel (2%) image showing amplified products
for TAP2 gene for position 565.
Lane 1 - 100bp molecular marker.
Lanes 2,3,4,5 and 6 - homozygous for Alanine/Alanine(GG).
Lane 7 - heterozygous for Alanine/Threonine (GA).
Internal control (400bp) is common for all PCR products.
166 TAP polymorphisms in leprosy
Table 6.5 : Genotype and Allele distribution of TAP2 (565) gene
polymorphism.
Table 6.6: Genotype and Allele distribution of TAP2(565) gene across
leprosy spectrum
Using the Fisher’s exact test the distribution of TAP2 565 genotypes
and alleles were compared. % represents the percentage frequency of
genotype; *p-value<0.05 was considered significant; OR: Odds Ratio;